Vincent Homburger

An α40 subunit of a GTP-Binding protein immunologically related to G0 mediates a dopamine-induced decrease of Ca2+ current in snail neurons

Dopamine induces a decrease in voltage-dependent Ca2+ current in identified neurons of the snail H. aspersa. This effect is blocked by intracellular injection of activated B. pertussis toxin and of an affinity-purified antibody against the alpha subunit of bovine Go protein. The dopamine effect is mimicked by intracellular injection of mammalian alpha o. In snail nervous tissue, pertussis toxin ADP-ribosylates a single protein band on SDS gels, and this band is recognized in immunoblots by the anti-alpha o antibody. We propose that this is a 40 kd alpha subunit of a molluscan G protein immunologically related to alpha o and that it mediates the effect of dopamine on Ca2+ currents in identified snail neurons.

Presence of three pertussis toxin substrates and Goα immunoreactivity in both plasma and granule membranes of chromaffin cells

GTP‐binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussis toxin is known to catalyze ADP‐ribosylation of several GTP‐binding proteins. In this paper, the subcellular localization of B. pertussis toxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP‐ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of Giα. Significant immunoreactivity with anti‐Goα antibodies was detected at the level of the 39 kDa faster component. The association of G‐proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.

Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

Synaptic activity–dependent remodeling of the glutamate receptor scaffold complex generates a negative feedback loop that limits further NMDA receptor activation.

Shank3-Rich2 Interaction Regulates AMPA Receptor Recycling and Synaptic Long-Term Potentiation

Synaptic long-term potentiation (LTP) is a key mechanism involved in learning and memory, and its alteration is associated with mental disorders. Shank3 is a major postsynaptic scaffolding protein that orchestrates dendritic spine morphogenesis, and mutations of this protein lead to mental retardation and autism spectrum disorders. In the present study we investigated the role of a new Shank3-associated protein in LTP. We identified the Rho-GAP interacting CIP4 homolog 2 (Rich2) as a new Shank3 partner by proteomic screen. Using single-cell bioluminescence resonance energy transfer microscopy, we found that Rich2-Shank3 interaction is increased in dendritic spines of mouse cultured hippocampal neurons during LTP. We further characterized Rich2 as an endosomal recycling protein that controls AMPA receptor GluA1 subunit exocytosis and spine morphology. Knock-down of Rich2 with siRNA, or disruption of the Rich2-Shank3 complex using an interfering mimetic peptide, inhibited the dendritic spine enlargement and the increase in GluA1 subunit exocytosis typical of LTP. These results identify Rich2-Shank3 as a new postsynaptic protein complex involved in synaptic plasticity.

Secretion of Protease Nexin-1 by C6 Glioma Cells Is under the Control of a Heterotrimeric G Protein, Go1

Heterotrimeric Go proteins have recently been described as regulators of vesicular traffic. The Goα gene encodes, by alternative splicing, two Goα polypeptides, Go1α and Go2α. By immunofluorescence and electron microscopy, we detected Go1α on the membrane of small intracellular vesicles in C6 glioma cells. After stable transfection of these cells, overexpression of Go1α but not Go2α was followed by a rise in the secretion of a serine protease inhibitor, protease nexin-1 (PN-1). This secretion was enhanced as a function of the amount of expressed Go1α. Metabolic cell labeling indicated that this increase in PN-1 secretion was not the result of an enhancement in PN-1 biosynthesis or a decrease in its uptake, but revealed a potential role of Go1α in the regulation of vesicular PN-1 trafficking. Furthermore, activators of Go proteins, mastoparan and a peptide derived from the amino terminus of the growth cone-associated protein GAP43, increased PN-1 secretion in parental and Go1α-overexpressing cells. Brefeldin A, an inhibitor of vesicular traffic, inhibited both basal and mastoparan-stimulated PN-1 secretions. These results indicate, that in C6 glioma cells, PN-1 secretion could be regulated by both Go1α expression and activation.

GTP binding proteins: a key role in cellular communication.

One of the major steps in the understanding of the hormonal and sensory transduction mechanisms in eukaryotic cells has been the discovery of a family of GTP binding proteins which couple receptors to specific cellular effectors. The absolute requirement of GTP for hormonal stimulation of adenylate cyclase was the initial observation which led to the purification of the protein involved: Gs. Gs couples stimulatory receptors to adenylate cyclase. It is a heterotrimer composed of an alpha chain (45 or 52 kDa), a beta chain (35-36 kDa) and a gamma chain (8 kDa). Several other G proteins of known functions have been purified: Gi, which couples inhibitory receptors to adenylate cyclase, and transducin which couples photoexcited rhodopsin to cyclic GMP phosphodiesterase. Some G proteins of uncertain function have also been purified: Go, a G protein mainly localized in nervous tissues and Gp, a G protein isolated from placenta and platelets. All these G proteins have a common design. Like Gs they all consist of 3 chains: alpha, beta and gamma. The beta chains are nearly identical, whereas the gamma chains are more variable. The alpha chains are different, but share common domains (especially at the level of the GTP binding site). These domains of homologies are also similar to those of other GTP binding proteins, such as the product of the ras gene (p21) and the initiation or elongation factors. alpha Chains are also ADP ribosylated by bacterial toxins. Gs and transducin are targets for cholera toxin, whereas Gi, Go and transducin are targets for pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuroblastoma Differentiation Involves the Expression of Two Isoforms of the α‐Subunit of Go

The regulation of GTP‐binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E‐115 cells. N1E‐115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to α‐subunits (Gα) of Go (a G protein of unknown function) and “Gi (a G protein inhibitory to adenylate cyclase)‐like” proteins and one substrate of Vibrio cholerae toxin corresponding to an α‐subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Goα was found, having a pI of 5.8. Goα content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Goα form having a pI of 5.55. Both Goα isoforms have similar sizes on sodium dodecyl sulfate‐polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Goα, are ADP‐ribosylated by PTX, and are covalently myristylated in whole N1E‐115 cells. In addition, immunofluorescent staining of N1E‐115 cells with Goα antibodies revealed that association of Goα with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Giα and Gsα did not significantly change, whereas that of the common β‐subunit increased by ∼ 30% over the same period. These results demonstrate specific regulation of the expression of Goα during neuronal differentiation.

EXPRESSION OF THE GUANINE NUCLEOTIDE-BINDING PROTEIN GO CORRELATES WITH THE STATE OF NEURAL COMPETENCE IN THE AMPHIBIAN EMBRYO

The nucleotide-binding protein Go is a transducing molecule closely associated with neural structures in vertebrates. Because of the potential importance of molecules of this type during the first step of neurogenesis, we have investigated the kinetics of expression of Go in the amphibian (Pleurodeles waltl) embryo, focusing our attention on the stages corresponding to the acquisition of neural competence by presumptive ectoderm and to the process of neural induction. Using affinity-purified IgGs directed against the alpha subunit of Go, Go-like immunoreaction (GoLI) is first detected at the midblastula stage in some animal cap (future ectodermal) cells just before they have attained competence to be neuralized. At the early gastrula stage, GoLI is almost exclusively expressed by neural-competent tissue as a whole, with no obvious difference between the dorsal (prospective neural) and the ventral (prospective epidermal) ectoderm. The expression of GoLI is therefore related to the state of competence of the tissue rather than to its fate. At the early neurula stage, immediately following neural induction, the expression of GoLI persists essentially in that part of ectoderm that has been diverted from epidermal differentiation towards the neural pathway; in the ventral ectoderm, as neural competence is lost GoLI disappears. Furthermore, in the neurectoderm, only approximately 70% of the cells conserve GoLI, demonstrating that immediately following neural induction the population of neurectodermal cells is not homogeneous.

Identification of Multiple Subunits of Heterotrimeric G Proteins on the Membrane of Secretory Granules in Rat Prolactin Anterior Pituitary Cells

The subcellular distribution of multiple subunits of heterotrimeric GTP-binding proteins has been investigated in rat anterior pituitary cells in primary culture, and more precisely in prolactin cells, by immunocytochemistry and subcellular fractionation followed by immunoblotting or ADP ribosylation, using polyclonal affinity-purified antibodies directed against Gi3 alpha, Gs alpha, Go1 alpha, Go2 alpha, and G beta. As expected, all these subunits were detected on the plasma membrane. They were, however, also detected on the membrane of several intracellular compartments involved in the secretory pathway, particularly on the secretory granule membrane. Differences appeared between the precise subcellular distribution and the local concentration of each subunit. The main subunits present on the secretory granule membrane were Gi3 alpha and Gs alpha. Go1 alpha, Go2 alpha, and G beta were detected, to a lesser extent, on parts of the membrane of a few secretory granules located near the plasma membrane. Domains of the rough endoplasmic reticulum cisternae were immunolabeled with anti-Gs alpha and anti-Go1 alpha. In the Golgi zone, the membrane of some vesicles was stained only with anti-Gs alpha and anti-Go2 alpha. The association of this set of heterotrimeric G protein subunits on the membrane of the secretory granules suggests that these subunits could be involved in the regulation of formation, storage, targeting, and/or exocytosis of these organelles.

Rho-GTPase-activating Protein Interacting with Cdc-42-interacting Protein 4 Homolog 2 (Rich2) A NEW Ras-RELATED C3 BOTULINUM TOXIN SUBSTRATE 1 (Rac1) GTPase-ACTIVATING PROTEIN THAT CONTROLS DENDRITIC SPINE MORPHOGENESIS

Background: Rich2 is a synaptic Rho-GAP (Rho-GTPase-activating protein) the target of which was unknown. Results: We found that Rich2 controls dendritic spine morphogenesis by inhibiting Rac1 activity. Conclusion: Rac1 is the target of Rich2 in spines. Significance: We identified for the first time Rich2 as a Rac1-GAP protein that plays an important role in spine formation. Development of dendritic spines is important for synaptic function, and alteration in spine morphogenesis is often associated with mental disorders. Rich2 was an uncharacterized Rho-GAP protein. Here we searched for a role of this protein in spine morphogenesis. We found that it is enriched in dendritic spines of cultured hippocampal pyramidal neurons during early stages of development. Rich2 specifically stimulated the Rac1 GTPase in these neurons. Inhibition of Rac1 by EHT 1864 increased the size and decreased the density of dendritic spines. Similarly, Rich2 overexpression increased the size and decreased the density of dendritic spines, whereas knock-down of the protein by specific si-RNA decreased both size and density of spines. The morphological changes were reflected by the increased amplitude and decreased frequency of miniature EPSCs induced by Rich2 overexpression, while si-RNA treatment decreased both amplitude and frequency of these events. Finally, treatment of neurons with EHT 1864 rescued the phenotype induced by Rich2 knock-down. These results suggested that Rich2 controls dendritic spine morphogenesis and function via inhibition of Rac1.